Hormones regulating the concentrations of estrogen (E) and oxytocin (OT) receptors will be studied with uterine and mammary explants maintained in organ culture. Cycloheximide is added to determine whether protein synthesis is necessary for the induction or maintenance of OT receptor concentrations in vitro. The relationship between the concentrations of E and OT receptors will be examined in the explant systems. OT-induced changes in phosphoprotein labeling in myometrial and myoepithelial cells preincubated with 32P are determined by two-dimensional gel electrophoresis and autoradiography. We will compare the dose-response relationship between phosphorylation changes and receptor binding for OT and several analogues; and determine whether the changes in phosphorylation are due to activation of kinases or phosphatases. Myometrial tubulins will be purified to confirm their identity as specific OT-modified phosphoproteins. OT inhibition of (Ca-Mg)ATPase from myometrial cell membranes will be studied. The Km values for ATP, Ca and Mg will be determined. The concentrations of OT and analogues giving half-maximal inhibition will be compared to their relative receptor affinities. To determine whether the (Ca-Mg)ATPase is a calcium pump, we will study the effects of OT and analogues on 45Ca efflux from plasma membrane vesicles. We hope to isolate the phosphoenzyme intermediate of (Ca-Mg)ATPase so that the molecular sites of action of Ca, Mg and OT can be determined by their effects on the formation and/or breakdown of phosphoenzyme. OT will be cross-linked to putative receptor sites with disuccinimidyl suberate. The apparent Mr and subunit structure of the cross-linked complex will be determined by SDS-gel electrophoresis and fluorography. Purification of triton solubilized complex will be attempted by immunoaffinity chromatography with antibodies to OT and by conventional protein purification methods.